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c. Inoculation and incubation Section III: Drug Susceptibility Testing • Label two MGIT PZA tubes, one as GC (growth control) and one as PZA (drug containing). Using a pipette, aseptically add 0.8 ml of PZA supplement to each of the two tubes. • Aseptically add 0.1 ml (100 µL) of the reconstituted drug into the PZA tube. If possible, use a micropipette. Try to be as accurate as possible in adding the drug. This will give you 100 µg PZA per ml of the medium. Do not add drug to the GC tube. • Inoculate 0.5 ml of the culture suspension to the PZA tube using a sterile pipette. • For growth control inoculation, first dilute the inoculum 1:10 by adding 0.5 ml of the culture suspension (the one used for the drug tube) to 4.5 ml of sterile saline. Mix well by tightening the cap and inverting at least 5-6 times. Add 0.5 ml of this diluted suspension into the tube labeled GC. Note: For PZA susceptibility test, the inoculum for the control is diluted 1:10 and not 1:100 as in SIRE AST). • Tighten the caps and gently invert both the MGIT tubes several times to mix. • Place them in a two AST Set Carriers (refer to the BACTEC MGIT 960 User’s Manual for further information) with the sequence of first GC and the PZA. • Enter the PZA set into the instrument using AST set entry feature (refer to the BACTEC MGIT 960 User’s Manual for further information). Make sure the GC is placed first, and PZA second, in the AST Set Carrier. Select PZA as the drug in 2nd tube AST set carrier definition when performing the AST set entry. • Check the purity of the inoculum by streaking a loopful of the culture suspension onto blood agar plate. If a blood agar plate is not available use Chocolate agar BHI agar. Incubate and check for growth after 48 hours. If growth appears on the streaked plate, discontinue the susceptibility test and do not use results of this susceptibility test. Repeat testing after obtaining a pure culture. Precautions: All the additions and handling of cultures should be done only inside a biosafety cabinet. To avoid contamination, use properly sterilized tubes, reagents and other items. Proper reconstitution of PZA drug and accurate addition of the drug to the medium is essential for getting correct results. Preparation of inoculum is critical. It should be as homogeneous as possible with the least amount of mycobacterial clumps. Dilution (1:10) of the culture suspension for the growth control and mixing is critical. Use only “PZA Supplement” and PZA medium for the PZA susceptibility test. Make sure the tubes in the AST set carrier are placed in the proper sequence (i.e. GC, PZA). MGIT TM Procedure Manual 50
5. Results Section III: Drug Susceptibility Testing The BACTEC 960 instrument will monitor the inoculated media and will give results within 4-21 days (Growth Control reaches GU 400 or more) once the test is complete. At this point, the susceptibility set can be removed after scanning and a report can be printed. The susceptibility report will be “S” (susceptible) or “R” (resistant). If the GC tubes become positive in less than 4 days or remain negative up to 21 days, or if some other conditions occur which may affect the test results, the instrument report will show an Error (“X”). In such situations, the test needs to be repeated. The instrument interpretation of results is based on GU values as described for SIRE drugs (Section III-A-5). 6. Reporting Report results as susceptible or resistant, indicating the method and concentration of the drug used. Mono-resistance to PZA is uncommon. In case mono-resistance is observed with a clinical isolate, repeat the test and report results only when it is confirmed. Cultures which are contaminated, or belong to an NTM species, or are a mixed culture of M. tuberculosis and other mycobacteria, will give erroneous results. Strains of M. bovis, including M. bovis BCG, are also naturally resistant to PZA. 7. Quality control It is extremely important to periodically perform a quality control of drug susceptibility testing for PZA. The minimum requirement is to test each new batch of reagents, such as PZA drug or MGIT PZA medium. If the QC batch fails, all the results obtained within that batch, as well as the new batch of a reagent, should be thoroughly reviewed and the testing should be repeated. Use M. tuberculosis H37Rv (ATCC [American Type Culture Collection] number 27294) as a QC strain which is susceptible to all anti-tuberculosis drugs. It is not necessary to include a resistant strain, as most of the resistant strains against a drug which are available from ATCC and other culture collections are highly resistant and do not give any added benefit in the quality control. The test procedure for QC organisms is the same as described above for clinical isolates. The inoculum could be from a freshly grown culture in the MGIT medium or on a LJ slant. If the suspension of QC bacteria is made from growth on solid medium, follow the procedure for suspension preparation as described above. The suspension may be stored in aliquots frozen, for up to six months, at -70ºC + 10ºC (for details of QC strain preparation, see Section II-K-2). If the QC batch fails, that is, the pan-susceptible H3Rv shows some resistance, then all the results obtained within that batch become invalid and the testing should be repeated. MGIT TM Procedure Manual 51
Page 1: Prepared for the Foundation for Inn
Page 4 and 5: TABLE OF CONTENTS F. Preparation an
Page 6 and 7: Section V: Appendices TABLE OF CONT
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Page 10 and 11: Section I: Principle of Procedures
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Page 14 and 15: Section II: Procedure for Primary I
Page 20 and 21: f. Urine Section II: Procedure for
Page 22 and 23: . Kinyoun’s staining Section II:
Page 24 and 25: 4. Reporting Section II: Procedure
Page 26 and 27: 2. Procedures a. Reconstituting PAN
Page 28 and 29: f. Detection of positive growth Sec
Page 30 and 31: 2. Dealing with contamination Secti
Page 38 and 39: d. Inoculation/incubation Section I
Page 40 and 41: 4. Record keeping Section II: Proce
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Page 46 and 47: 5. Testing at higher drug concentra
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Page 62 and 63: 2. Supplies for AFB smears 212522 T
Page 64 and 65: 3. Supplies for drug susceptibility
Page 66 and 67: Procedure for Ordering Cultures App
Page 68 and 69: c. Auramine O fluorescent (fluoroch
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Page 72 and 73: Appendix C • Do not use reagents
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Page 82 and 83: Appendix D � The personnel who wo
Page 84 and 85: Precautions Appendix D • Use a pu
Page 86 and 87: h. Interpretation of results Append
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